Cation chelator hot start

ABSTRACT

The invention is in the field of regulation of enzymatic activity in nucleic acid modifying reactions. It describes a method of regulating enzymatic activity by adding chelating agents to the reaction composition and exploits the fact that both the binding of divalent cations to these chelating agents and the pH of commonly used buffers is temperature dependent. PCR experiments that are hampered by non-specific side products can be regulated such that the target sequence is amplified in a more specific manner.

FIELD OF THE INVENTION

The present invention relates to the field of nucleic acid chemistry.More particular, it relates to the regulation of enzyme activity in thefield of nucleic acid modifying reactions.

BACKGROUND

Nucleic acid modifying reactions play a pivotal role in modernbiological and pharmaceutical research, both in the academic andindustrial settings. Such reactions cover a wide range of applications,ranging from nucleic acid amplification reactions to regulated andspecific cleavage of nucleic acids. These are mediated by enzymes thathave been studied extensively for the past decades.

Amplification of target nucleic acid sequences is of importance tomodern biological and pharmaceutical industry. Large-scale roboticfacilities used in industrial research depend on the accurate andefficient regulation of amplification conditions to ensure that thetarget sequences are correctly amplified for downstream applications.

Regulation of the activity of such enzymes is however not a trivialtask. In the case of polymerases, efficient amplification is dependenton a complex interplay of parameters such as primer length, GC contentof both primer and target sequences as well as ionic strength andcomposition of the reaction buffer. Further, non-specific binding ofprimers is often observed at lower temperatures during the amplificationcycles. This increases the fraction of non-specific side products andlowers the overall efficiency of the amplification reaction.

To address this, recent developments in the field of polymerasesdescribe “Hot Start” polymerases. This class of enzymes is eitherchemically inactivated or has the active site blocked due to binding ofa specific antibody or an aptamer. After an activation step at hightemperature, the chemical modification is cleaved off and the enzyme isactivated.

In addition to “Hot Start” polymerases, so called “Hot Start” primersand “Hot Start” nucleotides have also been developed. These arechemically modified primers, wherein the modification is cleaved off athigh temperatures and thus the primer is rendered functional and is ableto hybridize to its target sequence. However, synthesis of such primersis expensive and requires more time than standard primers. Both in thecase of the “Hot Start” primer and polymerases, the blocking featuresare only available once since the heat-induced removal of the chemicalmodification is irreversible.

There is a need for methods that allow for the regulation of enzymaticactivity without elaborate modification of enzymes and substrates.

BRIEF DESCRIPTION OF THE INVENTION

The present invention relates to a method of regulation of enzymaticactivity by controlling the concentration of divalent cations in thereaction composition.

The reaction composition comprises at least one enzyme, wherein theactivity of said enzyme is dependent on divalent cations; a chelatingagent; a divalent cation, wherein the binding of said cation to saidchelating agent is dependent on pH and/or temperature of the reactioncomposition; a buffering system, wherein the acid dissociation constantis temperature dependent, such that a change in temperature results in achange of pH of the aqueous solution; and a substrate of said enzyme. Inaddition, changing the temperature in the reaction composition resultsin divalent cations which are bound to chelating agents being releasedfrom these complexes and thereby the enzyme is activated or increasesactivity. Chelating said cation does not have structural consequences;selectively complexing said cation modulates activity. The change inactivity is reversible; inactivation by chelating can be reversed byreleasing said cation upon temperature increase.

The invention also relates to a kit for performing a nucleic acidmodifying reaction and comprises a buffer system, a chelating agent, anucleic acid modifying enzyme and a divalent cation for said enzyme.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method for the regulation of enzymeactivity in a reaction composition. The reaction composition comprisesat least one enzyme, wherein the activity of said enzyme is dependent ondivalent cations; a chelating agent; a divalent cation, wherein thebinding of said cation to said chelating agent is dependent on pH and/ortemperature of the reaction composition; a buffering system, wherein theacid dissociation constant is temperature dependent, such that a changein temperature results in a change of pH of the aqueous solution; and asubstrate of said enzyme. In addition, changing the temperature in thereaction composition results in divalent cations which are bound tochelating agents being released from these complexes and thereby theenzyme is activated or increases activity. Chelating said cation doesnot have structural consequences; selectively complexing said cationmodulates activity. The change in activity is reversible; inactivationby chelating can be reversed by releasing said cation upon temperatureincrease.

In a preferred embodiment, the present invention relates to a method forthe regulation of enzyme activity in a reaction composition. Thereaction composition comprises at least one enzyme, wherein the activityof said enzyme is dependent on divalent cations; a chelating agent; adivalent cation, wherein the binding of said cation to said chelatingagent is dependent on pH of the reaction composition; a bufferingsystem, wherein the acid dissociation constant is temperature dependent,such that a change in temperature results in a change of pH of theaqueous solution; and a substrate of said enzyme. In addition, changingthe temperature in the reaction composition results in divalent cationswhich are bound to chelating agents being released from these complexesand thereby the enzyme is activated or increases activity. Chelatingsaid cation does not have structural consequences; selectivelycomplexing said cation modulates activity. The change in activity isreversible; inactivation by chelating can be reversed by releasing saidcation upon temperature increase.

In one embodiment of the invention the enzyme is a nucleic acidmodifying enzyme.

In one embodiment, the activity of the nucleic acid modifying enzymecomprises substrate binding and substrate processing activity.

In a preferred embodiment the nucleic acid modifying enzyme is selectedfrom the group of polymerases, transcriptases and cation-dependentnucleases.

In a more preferred embodiment the polymerase is selected from the groupof organisms comprising Thermus, Aquifex, Thermotoga, Thermocridis,Hydrogenobacter, Thermosynchecoccus, Thermoanaerobacter, Pyrococcales,Thermococcus, Bacilus, Sulfolobus and non-thermophiles. Preferably theviral reverse transcriptases are from MMLV, AMV HIV, EIAV and/or thenuclease is a bovine DNase.

In the most preferred embodiment the polymerase is selected from thegroup of organisms comprising Aquifex aeolicus, Aquifex pyogenes,Thermus thermophilus, Thermus aquaticus, Thermotoga neopolitana, Thermuspacificus, Thermus eggertssonii and Thermotoga maritima.

In particular, the invention also describes a method, wherein theremoval of said divalent cation results in decreased or loss of activityof said nucleic acid modifying enzyme.

This represents an option to regulate enzymatic activity and substratebinding is at the level of the concentration of divalent cations in thereaction composition. For instance, in the case of polymerases and manynucleases, the concentration of divalent ions such as magnesium, calciumand others is crucial to the activity of the enzyme. A reduced level ofsaid cations leads to vastly decreased activity or even abolishesenzymatic activity. In the case of polymerases, stability ofhybridization of the primers to the target sequence is greatly reduced.Many nucleases possess a divalent cation in the active site that iscrucial to substrate processing. A way to regulate enzymatic activity onthe level of ion concentration exploits the fact that both the pH valueof buffers routinely used in enzymatic reaction mixtures and the abilityof chelating agents to bind ions is temperature-dependent. In additionto polymerases, other nucleic acid modifying enzymes such as nucleasesalso depend on divalent ions in their active site and therefore can beregulated as described above.

The invention also relates to a method, wherein the activity of saidnucleic acid modifying enzyme is selected from the group comprisingamplification, reverse transcription, isothermal amplification,sequencing and hydrolytic cleavage of ester bonds, preferablyamplification, reverse transcription, and hydrolytic cleavage of esterbonds.

In a preferred embodiment of the invention the chelating agent isselected from the group comprising ethylene di amine tetra acetic acid(EDTA), ethylene glycol bis(amino ethyl) N,N′-tetra acetic acid andnitrile acetic acid (NTA). Particularly preferred is EGTA.

In one embodiment the divalent cation is selected from the groupcomprising Mg²⁺, Ca²⁺, Mn²⁺, Cu²⁺, Fe²⁺, Ni²⁺, Zn²⁺ and Co²⁺. In apreferred embodiment, the chelating agent is EDTA and the cations areselected from Mg²⁺, Ca²⁺, Mn²⁺, Cu²⁺, Ni²⁺, Zn²⁺ and/or Co²⁺.

In one embodiment, several cations selected from the group comprisingMg²⁺, Ca²⁺, Mn²⁺, Cu²⁺, Fe²⁺, Ni²⁺, Zn²⁺and Co²⁺ are present in thereaction.

In one embodiment the chelating agent is EGTA and the cations are Ca²⁺and/or Mg²⁺.

In one embodiment the chelating agent is NTA and the cations are Ca²⁺and/or Cu²⁺and/or Co²⁺.

In another embodiment of the invention the buffer is suitable forenzymatic reactions. Preferably the buffer is selected from Table 4.Tris buffer is used in enzymatic reactions, preferably in PCRexperiments. The pH value of Tris buffer is temperature dependent. Atroom temperature, the pH is around 8.7. A shift in pH of 0.03 pH unitsper ° C. is observed. Therefore, at 95° C. the pH is 6.6.

In one embodiment, the concentration of the buffer system is between0.01 and 100 mM, preferably between 0.1 and 50 mM, more preferablybetween 1 and 30 mM and most preferably between 5 and 15 mM.

In one embodiment, the concentration of the divalent cation in thereaction is between 0.01 and 20 mM, preferably between 0.1 and 10 mM,most preferable between 1 and 8 mM.

In one embodiment, the concentration of the chelating agent is between0.05 and 50 mM, more preferably between 0.1 and 20 mM, even morepreferably between 0.5 and 10 mM, and most preferably between 1 and 8mM.

In one embodiment, the pH varies during the reaction in response to thetemperature change by at least 0.05 pH units, preferably by at least0.1, more preferably by at least 0.5, even more preferably by at least 1and most preferably by at least 2 pH units.

The invention relates to a method, wherein the reaction compositioncomprises a buffer system, preferably a Tris buffer system, wherein thedivalent cation is Mg²⁺, preferably at a concentration between 0.01 and20 mM; wherein the chelating agent is EGTA at a concentration between0.05 and 50 mM and wherein the nucleic acid modifying enzyme is a DNApolymerase, preferably a hot start polymerase. Preferred EGTAconcentration is between 0.1 mM and 20 mM, more preferred between 0.5 mMand 10 mM.

The invention also relates to a method, wherein the reaction compositioncomprises a buffer system, preferably a Tris buffer system; wherein thedivalent cation is selected from the group of Mg²⁺, Ca²⁺, Mn²⁺, Cu²⁺,Fe²⁺, Ni²⁺, Zn²⁺and Co²⁺; wherein the chelating agent is selected fromthe group of EGTA, EDTA and NTA and wherein the nucleic acid enzyme is anuclease.

Further, the invention relates to a kit for performing a nucleic acidmodifying reaction comprising a buffer system, a chelating agent, anucleic acid modifying enzyme and a divalent cation for said enzyme.

EXAMPLES

Selection of Chelating Agent

Tris buffer is routinely used in PCR buffers. At room temperature the pHof a Tris based PCR buffer is 8.7. Tris shows a temperature-dependentshift in pH value of 0.03 pH units per ° C. This means that at 95° C.the pH value is 6.6. In order to select a chelating agent for PCRexperiments, the pH-dependency of the binding constants of threedifferent chelating agents, NTA; EDTA and EGTA, was investigated. KnownpK values from literature for every chelating agent were used todetermine the pH dependency of the complex formation (FIG. 1).Thecorrelation curve for EGTA shows a strong correlation between pH valueand binding constant. Therefore, EGTA was selected for subsequentexperiments.

Endpoint PCR

An amplification experiment was performed using a test system that isknown to be prone to produce non-specific side products. A genomic DNAsequence of 1.2 kb was the target sequence. Primers HugA and HugB wereused. The primer sequences are as follows:

TABLE 1 Primer sequences used for endpoint PCR experiment. SEQ ID NOPrimer name Sequence 1 HugA CACACAGCGATGGCAGCTATGC 2 HugBCCCAGTGATGGGCCAGCT

Reactions with and without EGTA were performed in parallel. In set A,the magnesium concentration was varied in 1 mM steps, start and endpoint were 5 and 10 mM respectively. In set B, the start point was 0.5mM and the end point was 4 mM Mg. The setup is described in Table 2.

TABLE 2 HugI PCR reactions mixture. MM A MM B Puffer (—Mg) 1 1 X Taq0.625 0.625 Units Primer HugA 0.5 0.5 μM Primer HugB 0.5 0.5 μM dNTPs0.2 0.2 mM gDNA 10 10 ng EGTA 5 0 mM

The amplification program was as follows (Table 3):

TABLE 3 Amplification program of HugI PCR. Time [min:sec] Temp [° C.]03:00 95 00:30 94 01:00 59 01:00 72 10:00 72 4

35 cycles were performed.

The analysis of the PCR reactions on the agarose gel (FIG. 2) shows thatthe reactions containing EGTA as a chelating agent are more specific asof having less side products compared to those reactions without EGTA.Other buffers routinely used in enzymatic reactions are listed in Table4.

TABLE 4 List of buffers commonly used in enzymatic reactions. BufferProduct Useful pH ID No. # Description Range CAS Number pKa 1 A3594 ACESBioPerformance Certified, ≧99.0% 6.1-7.5 7365-82-4 6.80 2 B4554 BESBioPerformance Certified, cell culture 6.4-7.8 10191-18-1 7.10 tested,≧99.0% 3 B4429 BIS-TRIS BioPerformance Certified, cell 5.8-7.2 6976-37-06.50 culture tested, suitable for insect cell culture, ≧98% 4 B4679BIS-TRIS propane BioPerformance 6.3-9.5 64431-96-5 6.80 Certified, cellculture tested, ≧99.0% 5 E0276 EPPS BioPerformance Certified, cell7.3-8.7 16052-06-5 8.00 culture tested, ≧99.5% (titration) 6 G3915Gly-Gly BioPerformance Certified, cell 7.5-8.9 556-50-3 8.20 culturetested, ≧99% 7 H4034 HEPES BioPerformance Certified, ≧99.5% 6.8-8.27365-45-9 7.50 (titration), cell culture tested 8 H3784 HEPES sodiumsalt BioPerformance 6.8-8.2 75277-39-3 7.50 Certified, suitable for cellculture, ≧99.5% 9 H3662 HEPES sodium salt solution 1M, 75277-39-3BioReagent, suitable for cell culture 10 H3537 HEPES solution 1M,BioReagent, suitable 6.8-8.2 7365-45-9 for cell culture, suitable formolecular biology, 0.2 μm filtered 11 M2933 MES hydrate BioPerformanceCertified, 5.5-6.7 4432-31-9 6.10 suitable for cell culture, ≧99.5%(anhydrous) 12 M3058 MES sodium salt BioPerformance 5.5-6.7 71119-23-86.10 Certified, suitable for cell culture 13 M1317 MES solution 1M,BioReagent, for 5.5-6.7 molecular biology, suitable for cell culture 14M3183 MOPS BioPerformance Certified, cell 6.5-7.9 1132-61-2 7.20 culturetested, ≧99.5% (titration) 15 M9024 MOPS sodium salt BioPerformance6.5-7.9 71119-22-7 7.20 Certified, suitable for cell culture, ≧99.5% 16M1442 MOPS solution BioReagent, 1M, for 6.5-7.9 molecular biology,suitable for cell culture 17 P1851 PIPES BioPerformance Certified,suitable 6.1-7.5 5625-37-6 6.80 for cell culture, ≧99% 18 P5493Phosphate buffered saline 10× concentrate, BioPerformance Certified,suitable for cell culture 19 P5368 Phosphate buffered salineBioPerformance Certified, pH 7.4 20 S6191 Sodium chloride BioPerformanceCertified, ≧99.5% 7647-14-5 (titration), Cell Culture Tested 21 S6566Sodium phosphate monobasic 7558-80-7 Biotechnology PerformanceCertified, Cell Culture Tested 22 T5316 TAPS BioPerformance Certified,cell 7.7-9.1 29915-38-6 8.40 culture tested, ≧99.5% (titration) 23 T5441TAPS sodium salt BioPerformance 7.7-9.1 91000-53-2 8.40 Certified,suitable for cell culture, ≧99% 24 T5691 TES BioPerformance Certified,cell culture 6.8-8.2 7365-44-8 7.50 tested, ≧99% (titration) 25 T5816Tricine BioPerformance Certified, cell 7.4-8.8 1389475.00 8.10 culturetested, ≧99% (titration) 26 T7193 Trizma ® Pre-set crystalsBioPerformance 7.0-9.0 Certified, pH 7.0, average Mw 154.8 27 T9943Trizma ® Pre-set crystals BioPerformance 7.0-9.0 Certified, pH 7.0,average Mw 154.8 28 T7443 Trizma ® Pre-set crystals BioPerformance7.0-9.0 Certified, pH 7.2, average Mw 153.8 29 T7693 Trizma ® Pre-setcrystals BioPerformance 7.0-9.0 Certified, pH 7.4, average Mw 151.6 30T0319 Trizma ® Pre-set crystals BioPerformance 7.0-9.0 Certified, pH7.4, average Mw 151.6 31 T7818 Trizma ® Pre-set crystals BioPerformance7.0-9.0 Certified, pH 7.5, average Mw 150.6 32 T7943 Trizma ® Pre-setcrystals BioPerformance 7.0-9.0 Certified, pH 7.6, average Mw 149.0 33T8068 Trizma ® Pre-set crystals BioPerformance 7.0-9.0 Certified, pH7.7, average Mw 147.6 34 T8193 Trizma ® Pre-set crystals BioPerformance7.0-9.0 Certified, pH 7.8, average Mw 145.8 35 T8443 Trizma ® Pre-setcrystals BioPerformance 7.0-9.0 Certified, pH 8.0, average Mw 141.8 36T0819 Trizma ® Pre-set crystals BioPerformance 7.0-9.0 Certified, pH8.0, average Mw 141.8 37 T8568 Trizma ® Pre-set crystals BioPerformance7.0-9.0 Certified, pH 8.1, average Mw 139.8 38 T8943 Trizma ® Pre-setcrystals BioPerformance 7.0-9.0 Certified, pH 8.3, average Mw 135.4 39T8818 Trizma ® Pre-set crystals BioPerformance 7.0-9.0 Certified, pH8.5, average Mw 131.4 40 T1194 Trizma ® Pre-set crystals BioPerformance7.0-9.0 Certified, pH 8.5, average Mw 131.4 41 T9443 Trizma ® Pre-setcrystals BioPerformance 7.0-9.0 Certified, pH 8.8, average Mw 127.2 42T9568 Trizma ® Pre-set crystals BioPerformance 7.0-9.0 Certified, pH8.9, average Mw 125.6 43 T9693 Trizma ® Pre-set crystals BioPerformance7.0-9.0 Certified, pH 9.0, average Mw 124.6 44 T1444 Trizma ® Pre-setcrystals BioPerformance 7.0-9.0 Certified, pH 9.0, average Mw 124.6 45T9818 Trizma ® Pre-set crystals BioPerformance 7.0-9.0 Certified, pH9.1, average Mw 123.0 46 T0194 Trizma ® Pre-set crystals pH 7.2, average7.0-9.0 Mw 153.8 47 T0444 Trizma ® Pre-set crystals pH 7.5, average7.0-9.0 Mw 150.6 48 T0569 Trizma ® Pre-set crystals pH 7.6, average7.0-9.0 Mw 149.0 49 T0694 Trizma ® Pre-set crystals pH 7.7, average7.0-9.0 Mw 147.6 50 T0944 Trizma ® Pre-set crystals pH 8.1, average7.0-9.0 Mw 139.8 51 T1069 Trizma ® Pre-set crystals pH 8.3, average7.0-9.0 Mw 135.4 52 T1319 Trizma ® Pre-set crystals pH 8.8, average7.0-9.0 Mw 127.2 53 T6066 Trizma ® base BioPerformance Certified,41524.00 77-86-1 8.10 meets EP, USP testing specifications, cell culturetested, ≧99.9% (titration) 54 T5941 Trizma ® hydrochlorideBioPerformance 7.0-9.0 1185-53-1 8.10 Certified, cell culture tested,≧99.0% (titration) 55 T1819 Trizma ® hydrochloride solution pH 7.0,7.0-9.0 1M, BioReagent, for molecular biology, suitable for cell culture56 T2069 Trizma ® hydrochloride solution pH 7.2, 7.0-9.0 1M, BioReagent,for molecular biology, suitable for cell culture 57 T1944 Trizma ®hydrochloride solution pH 7.4, 7.0-9.0 0.1M 58 T2194 Trizma ®hydrochloride solution pH 7.4, 7.0-9.0 1M, BioReagent, for molecularbiology, suitable for cell culture 59 T2319 Trizma ® hydrochloridesolution pH 7.5, 7.0-9.0 1M, BioReagent, for molecular biology, suitablefor cell culture 60 T2944 Trizma ® hydrochloride solution pH 7.5,7.0-9.0 2M, BioReagent, for molecular biology, suitable for cell culture61 T2444 Trizma ® hydrochloride solution pH 7.6, 7.0-9.0 1M, BioReagent,for molecular biology, suitable for cell culture 62 T2569 Trizma ®hydrochloride solution pH 7.8, 7.0-9.0 1M, BioReagent, for molecularbiology, suitable for cell culture 63 T2694 Trizma ® hydrochloridesolution pH 8.0, 7.0-9.0 1M, BioReagent, for molecular biology, suitablefor cell culture 64 T3069 Trizma ® hydrochloride solution pH 8.0,7.0-9.0 2M, BioReagent, for molecular biology, suitable for cell culture65 T2819 Trizma ® hydrochloride solution pH 9.0, 7.0-9.0 1M, BioReagent,for molecular biology, suitable for cell culture

Modulation of Residual Activity of Chemically Inactivated Taq DNAPolymerase Using EGTA/EDTA

The following experiments employed a system to detect the formation ofprimer dimers in a PCR reaction mixture using residual active Taqpolymerase molecules. Herein, bisulphite-treated DNA is used as atemplate. As a consequence of the bisulphite treatment, which entailsthe chemical modification of non-methylated cytosines to uracil), saidtemplate only consists of three bases. Since bisulphite treatment onlyworks when using single stranded DNA, the majority of DNA aftercompletion of said bisulphite treatment is single stranded. Primers thatare used for amplification of such DNA sequences are characterized byreduced complexity since they only consist of three bases. Hence theseprimers are prone to dimer formation and are very likely to be able tobind >100.000 times to said bisulphite-treated DNA.

Genomic DNA was propagated using the Qiagen REPLI g Midi Kit accordingto the manufacturer's protocol. Subsequently, 1 μg of said genomic DNAwas used in 10 independent reactions wherein the DNA was subjected tobisulphite treatment using the EpiTect Bisulfite Kit followed bypurification. The resulting DNA of each reaction was pooled and used inthe subsequent amplification reactions. Primer sequences are shown inTable 5.

TABLE 5 Primers used in amplification of bisulphite-treated DNA. SEQPrimer ID NO NO Sequence 3 1 ACCCCCACTAAACATACCCTTATTCT 4 2GGGAGGGTAATGAAGTTGAGTTTAGG

TABLE 6 Amplification reaction mixture. Reagent Concentration Volume(μl) EpiTect HRM PCR Kit 2x 12.5 Primer 1 10 μM 1.875 Primer 2 10 μM1.875 Bisulphite-treated DNA 10 ng/μl 1 Water or EGTA 1-x mM 5 Water2.75

The final EGTA concentration was between 0.25 and 10 mM.

One set of samples consisting of two reactions was incubated on ice for120 min, whereas the other set of samples also consisting of tworeactions was incubated at room temperature for 120 min. Subsequentlyboth sets of samples were analyzed using the Rotor-Gene Q 5plex HRMSystem. The cycling program is shown in Table 7.

TABLE 7 Cycling program used in the amplification of bisulphite-treatedDNA. 95° C. - 5′ 95° C. - 10″ 55° C. - 30″ {close oversize bracket} x 4072° C. - 10″ HRM 68° C.-82° C.

Ct values are summarized in Table 8 and FIG. 3 shows the respectivemelting curves.

TABLE 8 Summary of results obtained from the amplification experiment ofbisulphite-treated DNA in the presence of different EGTA concentrations.Room temperature On ice EGTA [mM] Ct Average Standard deviaton CtAverage Standard deviation — 25.01 24.92 0.1 25.29 25.28 0.01 24.8225.26 0.25 25.2 25.31 0.11 25.34 25.43 0.09 25.42 25.51 0.5 25.55 25.510.04 25.53 25.58 0.04 25.47 25.62 0.75 25.91 25.86 0.05 25.78 25.83 0.0525.8 25.88 1 26.47 26.49 0.02 26.59 26.55 0.04 26.5 26.61 1.5 27.7627.82 0.06 28.17 28.19 0.02 27.88 28.21 2 29.9 29.81 0.09 30.16 29.350.82 29.71 28.53 4 37.32 37.13 0.1 38.37 38.26 0.11 37.02 38.14 8 1035.18 34.59 0.59 34

Samples that had been incubated on ice without the addition of EGTAshowed a Ct value of 25.28 and a specific melting curve (FIG. 3A)whereas the Ct value in the case of the samples that had been incubatedat room temperate is shifted to the right (24.92). In this case nospecific product was observed. Addition of EGTA up to a finalconcentration of 0.75 mM resulted in an increase of specificity withoutaffecting the Ct values. The amount of specific product increasedrapidly. In the case of EGTA concentrations exceeded 2 mM, successfulamplification was prevented (FIG. 3H).

In the follow-up experiment said primers and said bisulphite-treated DNAwere used in amplification reactions wherein the magnesium dependencywas analyzed. The composition of the reaction mixtures is shown in Table9.

TABLE 9 Amplification reaction mixture. Reagent Concentration Volume(μl) EpiTect HRM PCR Kit 2x 12.5 Primer 1 10 μM 1.875 Primer 2 10 μM1.875 Bisulphite-treated DNA 10 ng/μl 1 Water or EGTA 5 mM 2.5 Magnesium0.1 mM-0.6 mM 2.5 Water 2.75

The final concentration of EGTA was 5 mM. The HRM master mix wassupplemented with 0.1 0.6 mM magnesium.

Two sets of reactions consisting of duplicates were used in theamplification experiment. One set of samples was incubated on ice for120 min, whereas the other set of samples was incubated at roomtemperature for 120 min. Subsequently the samples were analyzed usingthe Rotor-Gene Q 5plex HRM System. The cycling program was the same asshown in Table 6. The results are shown in Table 9 and FIG. 4. Ct valuescorresponding to the samples incubated at room temperature and on icerespectively are shown in Table 10. FIG. 4 shows the respective meltingcurves.

TABLE 10 Summary of results obtained from the amplification experimentof bisulphite-treated DNA in the presence of EGTA and differentmagnesium concentrations. MgCl₂ Room temperature On ice DNA EGTA [mM] CtAverage Standard deviation Ct Average Standard deviation 10 ng — — 22.4122.62 0.21 25.32 25.46 0.14 22.82 25.59 5 mM 0 mM 28.86 28.77 0.09 28.7428.82 0.08 28.67 28.89 0.1 27.53 27.62 0.09 27.82 27.81 0.02 27.71 27.790.2 26.69 26.98 0.29 26.85 27.12 0.27 27.27 27.39 0.3 26.14 26.39 0.2526.25 26.38 0.13 26.64 26.51 0.4 26.06 26.15 0.09 26.01 26.08 0.06 26.2426.14 0.5 25.82 25.84 0.02 25.81 25.85 0.04 25.86 25.89 0.6 25.55 25.520.03 25.61 25.59 0.03 25.49 25.56

The experiment showed that in the case of the samples incubated on icewithout the addition of EGTA a Ct value of 25.46 and a specific meltingcurve was obtained, whereas incubation at room temperature resulted in ashift of the Ct value (22.62) and no specific amplification product wasobserved. Addition of EGTA up to a final concentration of 5 mM led toincreased specificity. The Ct value when using 5 mM EGTA was 28.77 and28.82 respectively. Increasing the magnesium concentration resulted inlower Ct values whilst maintaining specificity.

Modulation of DNase Activity

In this set of experiments means of modulating activity of DNase, anuclease isolated from bovine pancreas, were investigated.

Human genomic DNA was propagated using the REPLI g Midi Kit (Qiagen)according to the manufacturer's instructions. DNase activity wasanalyzed in 10 μl reactions. Each reaction contained 50 mM Tris pH 8.2as the reaction buffer, ˜1 μg genomic DNA, 1 mM MgCl₂, and 50 μM CaCl₂.Three different amounts of DNase (0.01, 0.1 and 1 U) were used. Thesamples were incubated at two different temperatures, 42° C. and on ice,for 5 and 15 min respectively. DNA degradation was terminated by addingEDTA to a final concentration of 8.33 mM and samples were incubated onice prior to analysis of the reaction products using a 0.5% agarose gel.The results are shown in FIG. 5. The gel shows that DNase is active onice. Reaction time and the amount of DNase strongly influencecompleteness of enzymatic digestion. Incubation of said genomic DNA onice for 15 min using 1 U DNase led to complete degradation of the sample(lane 3). Incubation at 42° C. led to complete degradation when usingany of the amounts of enzyme already after 5 min (lanes 8-13, ‘42° C’).

Addition of EGTA to a final concentration of 100 μM led to almostcomplete inhibition of degradation for any of the amounts of DNase thatwere used (lanes 2-7, ‘on ice’ ‘100 μM EGTA’). Exempt from this is thereaction using 1 U DNase for 15 min (lane 3 ‘on ice’ ‘100 μM EGTA’).However, in this case degradation is significantly reduced compared tothe sample without EGTA. Increasing the temperature to 42° C. largelyrestored DNase activity (lanes 8-13 ‘42° C.’ ‘100 μM EGTA’).

In the follow-up experiment EDTA was used as a chelating agent. Theprocedure of genomic DNA propagation as well the buffer and reactionconditions were equivalent to the experiment as described above.

The reaction products were analyzed using a 0.5% agarose gel (FIG. 6).Lanes 1-6 correspond to samples where the reaction was performed on ice.A comparison of lanes 1-3 and lanes 4-6 shows that addition of EDTAlargely reduced enzymatic activity. An increase in reaction temperatureto 42° C. results in bound Ca²⁺ ions being released from complexes withEDTA and thereby restoring enzymatic activity. Complete DNA degradationcan be observed in lanes 7-12.

In summary, both examples show that chelating agents can be used toinhibit DNase activity and that shifting the reaction temperaturerestores enzymatic activity, thereby validating said system of activityregulation.

FIGURE CAPTIONS

FIG. 1: Selection of chelating agents. pH-dependency of pK values ofEDTA, EGTA and NTA. Logarithmic values of the pK value was plottedversus the pH value.

FIG. 2: Agarose gel of Hugl PCR amplification experiment. The lanes areannotated as follows: 1: DNA ladder , 2: MMA +5 mM Mg, 3: MMA +6 mM Mg,4: MMA +7 mM Mg, 5: MMA +8 mM Mg, 6: MMA +9 mM Mg, 7: MMA +10 mM Mg, 8:DNA ladder, 9: DNA ladder, 10: MMB +0.5 mM Mg, 11: MMB +1 mM Mg, 12: MMB+2 mM Mg, 13: MMB +3 mM Mg, 14: MMB +4 mM Mg, 15: DNA ladder.

An increase in Mg concentration leads to successful amplification of thetarget product ‘specific PCR product’, although much unspecific productis visible (lane 10lane 12). A further increase of Mg concentrationleads to generation of unspecific PCR products (lane 14, 4 mM Mg Cl₂).In contrast, addition of 5 mM EGTA in presence of 5-10 mM MgCl₂ resultsin specific PCR product (lane 2-lane 7), although the amount ofunspecific by product increase while Mg concentration is increased.

FIG. 3

Melting curves (EGTA titration experiment).

The curves are annotated as follows: A: no additive, B: 0.25 mM EGTA, C:0.5 mM EGTA, D: 0.75 mM EGTA, E: 1 mM EGTA, F: 1.5 mM, G: 2 mM EGTA, H:4-10 mM.

FIG. 4

Melting curves (Mg titration experiment).

The curves are annotated as follows: A: no additives, B: 5 mM EGTA, C: 5mM EGTA+0, 1 mM Mg, D: 5 mM EGTA+0.2 mM Mg, E: 5 mM EGTA+0.3 mM Mg, F: 5mM EGTA+0.4 mM Mg, G: 5 mM EGTA+0.5 mM Mg, H: 5 mM EGTA +0.6 mM Mg.

FIG. 5

Agarose gel analysis of DNase assay at different temperatures andinfluence of EGTA.

Lanes are annotated as follows: Note that reactions corresponding tosamples in lane 2-7 were performed on ice and are hence labelled ‘onice’. Similarly, reactions corresponding to samples in lanes 8-13 wereperformed at 42° C. and are labelled ‘42° C.’ accordingly. Reactions atboth temperatures were performed in the absence and presence of 100 μMEGTA CO μM EGTA and 100 μM EGTA respectively).

Lane M: GelPilot High Range Ladder (60), lane 1: 1 μg WGA gDNA (no DNaseadded), lanes 2 and 3: 1 μl DNase (1U) 5 and 15 min, lanes 4 and 5: 0.1μl DNase (0.1U) 5 and 15 min, lanes 6 and 7: 0.01 μl DNase (0.01U), 5and 15 min, lanes 8 and 9: 1 μl DNase (1U) 5 and 15 min, lanes 10 and11: 0.1 μl DNase (0.1U) 5 and 15 min, lanes 12 and 13: 0.01 μl DNase(0.01U) 5 and 15 min.

FIG. 6

Agarose gel analysis of DNase assay at different temperatures andinfluence of EDTA.

Reactions corresponding to samples in lanes 1-6 were performed on iceand are hence labeled ‘on ice’ Reactions corresponding to samples inlane 7-12 were performed at 42° C. and are labeled ‘42 ° C.’accordingly. Lanes are annotated as follows: Lane K: 1 μg WGA gDNA.

Lane 1: 1 U DNase , lane 2: 0.5 U DNase , lane 3: 0.1 U DNase, Lane 4: 1U DNase +100 μM EDTA, lane 5: 0.5 U DNase +100 μM EDTA, lane 6: 0.1 UDNase +100 μ.M EDTA.

Lane 7: 1 U DNase, lane 8: 0.5 U DNase, lane 9: 0.1 U DNase, lane 10: 1U DNase +100 μM EDTA, lane 11: 0.5 U DNase +100 μM EDTA, lane 12: 0.1 UDNase +100 μM EDTA, lane M: GelPilot 1 kb Ladder (3 μl).

1. A method for regulation of enzyme activity in a reaction composition,comprising: (i) providing a reaction composition comprising: a. at leastone enzyme, wherein enzymatic activity of said enzyme depends onpresence of divalent cations in the reaction composition, b. one or aplurality of divalent cations, c. a chelating agent to which saiddivalent radon reversibly binds to form a complex, wherein binding ofsaid divalent cation to the chelating agent is dependent on either orboth of pH and temperature of the reaction composition, d. a bufferingsystem, having an acid dissociation constant that is temperaturedependent, such that a change in temperature in the reaction compositionresults in a change of pH of the reaction composition, and e. substratefor said enzyme; and (ii) changing the temperature in the reactioncomposition, such that the divalent cations which are reversibly boundto the chelating agents are released from the complex, wherein theenzyme is thereby activated or enzymatic activity of the enzyme isincreased relative to the activity prior to said step of changing thetemperature.
 2. The method of claim 1, wherein the enzyme is a nucleicacid modifying enzyme.
 3. The method of claim 2, wherein the nucleicacid modifying enzyme is selected from the group consisting ofpolymerases, reverse transcriptases and nucleases.
 4. The method ofclaim 2, wherein the enzymatic activity of said nucleic acid modifyingenzyme comprises substrate binding and substrate processing.
 5. Themethod of claim 2, wherein prior to changing the temperature, binding ofthe divalent cation to the chelating agent causes removal of saiddivalent cation from the nucleic acid modifying enzyme and results indecreased enzymatic activity or loss of enzymatic activity.
 6. Themethod of claim 1, wherein the chelating agent is selected from thegroup consisting of ethylene diamine tetra-acetate (EDTA), ethyleneglycol bis(amino ethyl)N,N′-tetra-acetate (EGTA) and nitrilo-tri-acetate(NTA).
 7. The method of claim 1, wherein the divalent cation is selectedfrom the group consisting of Mg²⁺, Ca²⁺, Mn²⁺, Cu²⁺, Fe²⁺, Ni²⁺, andCo²⁺.
 8. The method of claim 1, wherein the chelating agent is EDTA andthe divalent cation is selected from Mg²⁺, Ca²⁺, Mn²⁺, Cu²⁺, Zn²⁺ andCo²⁺.
 9. The method of claim 1, wherein the chelating agent is EGTA andthe divalent cation is either or both of Ca²⁺ and Mg²⁺.
 10. The methodof claim 1, wherein the chelating agent is NTA and the divalent cationsare one or more of Ca²⁺, Cu²⁺ and Co²⁺.
 11. The method of claim 2,wherein one or more of: (i) the reaction composition comprises a Trisbuffer system; (ii) the divalent cation is Mg²⁺ at a concentrationbetween 0.01 and 20 mM; (iii) chelating agent is EGTA at a concentrationbetween 0.05 and 50 mM; and (iv) the nucleic acid modifying enzyme is aDNA polymerase.
 12. The method of claim 2, wherein one or more of: (i)the reaction composition comprises a a Tris buffer system; (ii) thedivalent cation is selected from the group consisting of Mg²⁺, Ca²⁺,Mn²⁺, Cu²⁺, Fe²⁺, Ni²⁺, Zn²⁺ and Co²⁺; (iii) the chelating agent isselected from the group of EGTA, EDTA and NTA; and (iv) the nucleic acidmodifying enzyme is a nuclease.
 13. A kit for performing a nucleic acidmodifying reaction comprising: i. a buffer system; ii. a chelatingagent; iii. a nucleic acid modifying enzyme, iv. a divalent cation uponwhich enzymatic activity of said enzyme depends.
 14. The method of claim11 wherein the DNA polymerase is a hot start polymerase.